R21 application for Membrane Protein Production and Structure Determination (RFA-RM-04-026) Eph receptors, the largest subfamily of receptor tyrosine kinases, and their ephrin ligands are important mediators of cell-cell communication regulating cell attachment and mobility and Eph signaling is crucial for the development of many tissues including the nervous and cardiovascular systems. Both receptors and ligands are membrane-bound and their interactions at sites of cell-cell contact initiate unique bi-directional signaling cascades. To date there is no structural information on how extracellular ligand binding results in the activation of intracellular kinase domain in any receptor kinase. Research in this proposal focuses on the use of X-ray crystallography combined with other biophysical techniques and cell biology assays to study how Eph receptors are activated using full-length transmembrane molecules. Milligram amounts of a complete full-length EphA receptor will be expressed in mammalian cells or produced using a semisynthetic approach, purified and characterized. A new BIAcore-based assay will be developed for a rapid monitoring of a proper folding and enzymatic activity of the produced receptors. The structure of a full-length EphA receptor will then be determined alone and in a complex with a bound ligand using recent advances in the crystallization and structure determination of membrane proteins. The structures will elucidate the molecular events occurring during Eph-mediated signaling, namely the formation of higher-order receptor/ligand assemblies and the concomitant phosphorylation of the tyrosine residues of the kinase domain. We believe our studies will not only lead to the elucidation of the activation mechanism but will also provide new approaches for the crystallization of membrane proteins, and in particular single-pass cell surface receptors.